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MEDICAL SCIENCES
Detection and quantification of mutations in the plasma of patients with colorectal tumors
*Howard Hughes Medical Institute and The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, 1650 Orleans Street, Baltimore, MD 21231; and Indivumed, Center for Cancer Research at Israelitic Hospital, Orchideenstieg 14, 22297 Hamburg, Germany
Contributed by Bert Vogelstein, September 16, 2005
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Abstract |
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The early detection of cancers through analysis of circulating DNA could have a substantial impact on morbidity and mortality. To achieve this goal, it is essential to determine the number of mutant molecules present in the circulation of cancer patients and to develop methods that are sufficiently sensitive to detect these mutations. Using a modified version of a recently developed assay for this purpose, we found that patients with advanced colorectal cancers consistently contained mutant adenomatous polyposis coli (APC) DNA molecules in their plasma. The median number of APC DNA fragments in such patients was 47,800 per ml of plasma, of which 8% were mutant. Mutant APC molecules were also detected in >60% of patients with early, presumably curable colorectal cancers, at levels ranging from 0.01% to 1.7% of the total APC molecules. These results have implications for the mechanisms through which tumor DNA is released into the circulation and for diagnostic tests based on this phenomenon.
colorectal cancer | plasma DNA | tumor suppressor gene | circulating DNA | diagnosis
The discovery of the genetic bases of neoplasia has led to new approaches to detect tumors noninvasively (6–8). Several of these approaches rely on the ex vivo detection of mutant forms of the oncogenes and tumor suppressor genes that are responsible for the initiation and progression of tumors. This approach was first used to detect bladder and colon tumors through examination of urine and stool, respectively (9, 10), and has since been used to detect several other tumor types (11–14). Because the mutant genes are not only "markers" for cancer but also the proximate causes of tumor growth (1), they have major conceptual advantages over conventional markers such as fecal occult blood or serum PSA. In particular, conventional markers are not pathogenically involved in the tumorigenic process and are much less specific for neoplasia than are mutations.
The evaluation of patient blood samples for mutant DNA molecules is a particularly attractive approach because such tests could detect many different forms of cancers. Additionally, blood can be easily obtained from patients during routine outpatient visits, and methods for preparing and storing plasma and serum are well known and reliable. Accordingly, numerous studies have attempted to identify abnormal forms or quantities of DNA in plasma or serum (6, 11–15). Unfortunately, the results of many of these studies are contradictory. Some report high detection rates of cancers, and others report very low detection rates, despite the use of similar techniques and patient cohorts. Moreover, several studies have shown that loss of heterozygosity is routinely detectable in circulating DNA, even in patients with relatively nonaggressive tumors. To detect loss of heterozygosity in such samples, the neoplastic cells within a tumor must contribute >50% of the total circulating DNA.
The above studies, although promising, lead to several questions that must be answered to engender confidence in the use of circulating, abnormal DNA as a biomarker of malignancy. First, how many copies of a given gene fragment are present in the circulation in cancer patients? Second, what is the nature of this DNA (e.g., intact vs. degraded)? Third, what fraction of these gene fragments have an abnormal (e.g., mutant) DNA sequence? And, fourth, how does this fraction vary with stage of disease? To answer these questions, it was necessary to develop technologies that could simultaneously quantify the number of normal and mutant DNA molecules in a given sample, even when the fraction of mutant molecules was very small. In the current study, we employ such a technology to investigate circulating DNA in patients with colorectal tumors.
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Materials and Methods |
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Real-Time PCR. Primers were designed to generate 
100-bp amplicons that included one or more mutation sites. A universal tag (5'-TCCCGCGAAATTAATACGAC-3') was added to the 5' end of either the forward or reverse primer used to generate each amplicon. The sequences of these primers are listed in the supporting information. PCR was performed in 50-µl reactions containing 10 µl of 5x Phusion HF buffer, a 0.2 mM concentration of each dNTP, a 1 µM concentration of each primer, 1:50,000 dilution of SYBR green I (Invitrogen), 1.5 units of Phusion DNA polymerase (NEB, Beverly, MA), and 15 µl of purified plasma DNA (equivalent to 100 µl of plasma) or genomic DNA purified from normal mononuclear cells of the blood of healthy volunteers. The amplifications were carried out with an iCycler (Bio-Rad) under the following conditions: 98°C for 1 min; 98°C for 10 s, 70°C for 10 s, and 72°C for 10 s 3 times; 98°C for 10 s, 67°C for 10 s, and 72°C for 10 s 3 times; 98°C for 10 s, 64°C for 10 s, and72°C for10 s 3 times; and 98°C for 10 s, 61°C for 10 s, and 72°C for 10 s 30 times. Each reaction was performed in duplicate, and a calibration curve was generated in each 96-well plate by using various amounts of normal human genomic DNA. The concentration of PCR products was determined by using a PicoGreen dsDNA quantification assay (Invitrogen).
BEAMing. A common oligonucleotide (5'-TCCCGCGAAATTAATACGAC-3') was synthesized with a dual biotin group at the 5' end and with a six-carbon linker (C6) between the biotin and the other nucleotides (Integrated DNA Technologies, Coralville, IA). This oligonucleotide was coupled to streptavidin-coated magnetic beads (MyOne, Dynal, Oslo) according to the protocol described in ref. 16. The water-in-oil emulsions were prepared by modifications of the methods described by Ghadessy and Holliger (17) and Bernath et al. (18). For each emulsion PCR, a 240-µl aliquot of an aqueous PCR mix was added to 960 µl of 7% (wt/vol) Abil EM90 (Degussa Goldschmidt Chemical, Hopewell, VA) in mineral oil (Sigma). The aqueous phase contained 67 mM Tris·HCl (pH 8.8), 16.6 mM (NH4)2SO4, 6.7 mM MgCl2, 10 mM 2-mercaptoethanol, a 0.2 mM concentration of each dNTP, 0.05 µM forward primer (5'-TCCCGCGAAATTAATACGAC-3'), 8 µM reverse primer, 0.2 units/µl Platinum Taq polymerase (Invitrogen), 3 x 105 per µl oligonucleotide-coupled beads, and 0.1 pg/µl template DNA. The reverse primers are listed in the supporting information. The water–oil mix was vortexed for 10 s and then emulsified for 50 s by using an Ultra-Turrax homogenizer (T25 basic, IKA, Wilmington, NC) with a disposable OmniTip (Omni International, Waterbury, CT) at the minimum speed. The emulsions were aliquoted into 8 wells of a 96-well PCR plate and cycled under the following conditions: 94°C for 2 min; 94°C for 10 s, 58°C for 15 s, and 70°C for 15 s 50 times. After PCR, the emulsions were pooled into a 15-ml tube and demulsified through the addition of 10 ml of NX buffer (100 mM NaCl/1% Triton X-100/10 mM Tris·HCl, pH 7.5/1 mM EDTA/1% SDS). After vortexing for 10 s, the beads were pelleted by centrifugation for 5 min at 4,100 x g. The top phase was removed, and the beads were resuspended in 800 µl of NX buffer and transferred to a 1.5-ml tube. The beads were collected by using a magnet (MPC-S, Dynal) and washed with 800 µl of wash buffer (20 mM Tris·HCl,pH8.4/50 mM KCl). The double-stranded DNA on the beads was converted to single-stranded DNA by incubation in 800 µl of 0.1 M NaOH for 2 min at room temperature. The beads were washed twice with 800 µl of wash buffer, using the magnet, and finally resuspended in 200 µl of wash buffer. Single base extension and flow cytometry were performed as described in the supporting information.
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We conclude that the mutant DNA fragments present in the circulation of cancer patients are degraded compared with the circulating DNA derived from nonneoplastic cells. This conclusion is consistent with previous studies of other tumor types (21, 22) and has important implications for the detection of such mutant molecules.
Development of a Quantitative Assay for Detection of Rare Mutations. The results described above were obtained by sequencing hundreds of PCR products, each derived from one or a few DNA template molecules. In preliminary studies, we found that such digital PCR-based techniques were sufficiently sensitive to detect circulating mutant DNA molecules in patients with advanced cancers but not in patients with early stage cancers. To increase the sensitivity and reliability of these assays, we developed an extension of BEAMing (which derives its name from its principal components: beads, emulsion, amplification, and magnetics) that allowed us to examine many more template molecules in a convenient fashion. The approach consists of four steps. (i) Real-time PCR was used to determine the number of total APC gene fragments in the plasma sample (Fig. 2A, step 1). (ii) BEAMing was used to convert the amplified plasma DNA into a population of beads (Fig. 2A, steps 2–4). (iii) The mutational status of the extended beads was determined by single base extension (Fig. 2B). (iv) Flow cytometry was used to simultaneously measure the FITC, Cy5, and phycoerythrin (PE) signals of individual beads.
Fig. 3 shows a representative flow cytometry result wherein the interpretation of the profiles was confirmed experimentally. In the example shown, 342,573 beads were analyzed by flow cytometry. The single bead population (295,645) was used for the fluorescence analysis (Fig. 3A). Of these, 30,236 exhibited a PE signal (Fig. 3B), indicating that they had been extended during the emulsion PCR. The FITC and Cy5 signals reflected the number of beads containing mutant or WT sequences, respectively. Beads containing the WT DNA sequences (30,186) had high Cy5 but background FITC signal ("red beads" in Fig. 3C). Beads extended only with mutant DNA sequences (22) had high FITC signals but background Cy5 signals ("green beads"). Twenty-eight had both FITC and Cy5 signals ("blue beads"). Such dual-labeled beads resulted from either the presence of both a WT and mutant template in the droplet containing the bead or an error in the early cycles of the emulsion PCR (see below). These dual-labeled beads were eliminated from analysis, and only homogeneously labeled beads were considered for the enumeration of mutations. Note that this conservative analysis strategy results in a slight underestimation of the fraction of mutations, because it excludes mutants that were present in droplets that also contained one or more WT fragments. Beads in each of these three populations were collected by flow sorting, and single beads from the sort were used as templates in conventional DNA sequencing. All 131 beads subjected to sequencing analysis showed the expected patterns, with examples illustrated in Fig. 3C.
Limits to the Sensitivity of Assays for Plasma DNA Mutations. The results described above show that the BEAMing approach can, in principle, detect a very small fraction of fragments containing mutant sequences within a much larger pool of fragments containing WT sequence. Because >50 million beads are used in a single emulsion PCR and flow cytometry can be performed at speeds of >50,000 beads per s, the capacity to enumerate such mutations is not limited by the beads themselves. Instead, two other features limit the sensitivity. First, there is a finite number of DNA fragments present in clinical samples. As noted above, this number ranged from 1,350 to 230,000 fragments per ml in the patients with tumors (Table 1) and from 1,150 to 8,280 fragments per ml in control patients, which gives an upper bound to the sensitivity of the assays. For example, a calculation using the Poisson distribution shows that if 4,000 fragments were analyzed, the mutation fraction in circulating DNA would have to be >1 in 1,333 fragments (i.e., 3 divided by the number of total fragments analyzed) for the assay to achieve 95% sensitivity. A second limiting feature is the error rates of the polymerases used for PCR. In our approach, two PCR steps are used: The first is a conventional PCR that employs plasma DNA fragments as templates, and the second is an oil-in-water emulsion PCR that uses the initial PCR products as templates. In the emulsion PCR, errors occurring during the early rounds of PCR can result in heterogeneous beads containing both WT and mutant sequences. These beads are easily eliminated from consideration, as described in Fig. 3C. However, the errors introduced in the first PCR cannot be eliminated, because they give rise to beads with homogeneous mutant sequences, indistinguishable from those resulting from genuine mutations in the original plasma DNA templates.
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These issues suggest that the sensitivity of assays for circulating mutant DNA could be increased in the future by (i) the development of new or modified polymerases with reduced error rates and (ii) the use of more plasma per assay (i.e., more template molecules).
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A BEAMing assay was then designed for each of the mutations identified in the 33 tumors and applied to the DNA purified from the plasma of the corresponding patients (Table 1). In each case, DNA from normal lymphocytes or plasma from patients without cancer was used as a negative control. DNA from the tumors of the 33 patients was used as a positive control. All six patients with advanced lesions (Dukes' D, defined as having at least one distant metastatic lesion) were found to contain mutant DNA fragments in their plasma. Among 16 patients harboring cancers with a favorable prognosis (Dukes' A or B, defined as having no lymph node involvement and no distant metastases), 10 (63%) were found to contain mutant DNA fragments in their plasma. In contrast, among 11 patients with large, benign tumors (adenomas), only 1 patient's plasma was found to contain mutant DNA fragments. Representative flow cytometric results are shown in Fig. 4 and summarized in Table 1.
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Table 1 also lists the concentration of total APC fragments (WT plus mutant) in these patients' plasma. There was no direct relationship between the concentration of total APC fragments and the mutational load. Although patients with advanced cancers tended to have higher concentrations of total APC fragments than the other patients, this increase was not due to DNA from neoplastic cells. Furthermore, no correlation was found between tumor burden (volume of primary tumor plus metastatic sites) and either the concentration of APC fragments or percentage of mutant APC fragments in the circulation.
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But there are still a large number of mutant DNA fragments circulating in cancer patients. Assuming that the volume of distribution of DNA at steady state is similar to that of oligonucleotides in primates (60–70 ml/kg), an 8% fraction of mutant molecules among 47,800 fragments per ml of plasma (as in Dukes' D patients) would correspond to 1.6 x 107 mutant fragments present in a 70-kg person at any given time (24). The half-life of this tumor DNA is estimated at 16 min, based on the data obtained from clearance of fetal DNA in maternal plasma (25), which translates to 6 x 108 mutant fragments released from the tumor each day. For patients with a tumor load 100 g in size (3 x 1010 neoplastic cells), we thereby estimate that 3.3% of the tumor DNA is fed into the circulation on a daily basis. For a Dukes' B cancer of 30 g in which 1.3% of the 4,000 circulating APC fragments per ml of plasma are mutant, the corresponding estimate is that 0.15% of the tumor DNA is fed into the circulation each day.
So how do mutant APC gene fragments get into the plasma? Several clues are provided by our data. The ability to get into the circulation was clearly not related to tumor size, because the benign tumors we studied were as large as the cancers (Table 1), yet the former rarely gave rise to detectable mutant DNA fragments. Similarly, there was no significant correlation between the tumor load (including metastatic deposits) and the amount of mutant DNA in the circulation. In contrast, the degree of invasion was indeed correlated with the number of circulating DNA fragments. Those lesions that weren't invasive (benign tumors) did not commonly feed mutant DNA molecules into the plasma. As tumors invaded through more layers of the intestinal wall in Dukes' B vs. Dukes' A tumors, and through the intestine to distant sites in Dukes' D vs. Dukes' B tumors, the number of circulating mutant DNA molecules progressively increased (Fig. 5).
Another clue is provided by the size of the mutant DNA molecules. The data in Fig. 1 show that mutant sequences are enriched in small DNA fragments and could not be identified at all in fragments of 1,296 bp.
Based on these observations, we propose that the mutant DNA fragments found in the circulation are derived from necrotic neoplastic cells that had been engulfed by macrophages. As tumors enlarge and invade, they are more likely to outgrow their blood supply. Thus, invasive tumors generally contain large regions of necrosis, whereas benign tumors rarely do (26–29). Necrotic cells are not thought to release DNA into the extracellular milieu (30). However, cells that die from necrosis or apoptosis are routinely phagocytosed by macrophages or other scavenger cells. Interestingly, it has been shown that macrophages that engulf necrotic cells release digested DNA into the medium, whereas macrophages that engulf apoptotic cells do not (30). Moreover, the size of the DNA released from macrophages is small (30). All of these observations are consistent with a model wherein hypoxia induces necrosis of tumors, leading to the phagocytosis of tumor cells and the subsequent release of the digested DNA into the circulation. As tumors become more aggressive, the degree of this necrosis increases and the absolute amount of circulating mutant DNA correspondingly rises. Because necrosis involves the killing of neoplastic cells and surrounding stromal and inflammatory cells within the tumor, the DNA released from necrotic regions is likely to contain WT DNA sequences as well as mutant sequences. This phenomenon may explain the increase in total (nonmutant) circulating DNA observed in the plasma of patients with advanced cancers.
Clinical Implications. The ability to detect and quantify mutant DNA molecules in the circulation has obvious clinical importance, and this line of research has been pursued by several investigators. Our results inform the field in several ways. First, it is unlikely that circulating mutant DNA could be used to detect premalignant tumors, based on the fact that we were unable to detect such DNA even in very large adenomas. Second, it is unlikely that loss of heterozygosity detection or other techniques that require a majority of the circulating DNA to be derived from neoplastic cells will allow such detection, at least in colorectal cancers, because the proportion of mutant DNA fragments in plasma was small, averaging only 11% of the total DNA fragments even in large, metastatic cancers.
On the positive side, our data show that even relatively early cancers give rise to circulating mutant DNA fragments that can be detected with sufficiently sensitive and specific assays. In fact, >60% of cancers that had not yet metastasized gave rise to detectable mutant fragments in plasma. Even Dukes' A tumors, which are by definition barely invasive, were detectable with BEAMing-based assays. Virtually all Dukes' A tumors and most Dukes' B tumors can be cured with conventional surgery alone, without the need for adjuvant therapies (31).
In practical terms, plasma-based assays for mutant DNA fragments are inferior in several ways to more conventional techniques for early colorectal cancer detection. Colonoscopy is the gold standard, with sensitivity rates of >80% for adenomas and >90% for cancers (32). In particular, adenomas detected by colonoscopy can often be removed through the colonoscope, alleviating the need for surgery. Unfortunately, a variety of issues limits the widespread applicability of colonoscopy (either conventional or virtual) to the screening of asymptomatic patients (3, 5, 33), a fact that has stimulated the development of noninvasive technologies. One of the most promising of these noninvasive technologies is the analysis of fecal DNA for mutations (34). Because of the frequent presence of mutant DNA molecules in feces from both adenomas and early cancers, fecal DNA analysis is superior to plasma with regard to sensitivity. However, plasma-based assays have potential advantages with regard to ease of implementation and compliance.
For many tumor types, there are currently no alternative methods for presymptomatic diagnosis, unlike the case with colorectal cancers. In these other tumor types, the evaluation of circulating DNA could be particularly useful. Even if such assays could detect only a fraction of patients with treatable cancers, much morbidity and mortality could be averted.
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Acknowledgements |
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Footnotes |
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Conflict of interest statement: Under a licensing agreement between EXACT Sciences and The Johns Hopkins University, K.W.K. and B.V. are entitled to a share of royalties received by the university on sales of products related to digital PCR. Under a licensing agreement between Agencourt Biosciences Corporation and The Johns Hopkins University, D.D., K.W.K., and B.V. are entitled to a share of royalties received by the university on sales of products related to the use of BEAMing for preparing templates for DNA sequencing. The terms of these arrangements are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.
Abbreviations: APC, adenomatous polyposis coli; PE, phycoerythrin.
To whom correspondence should be addressed. E-mail: vogelbe{at}jhmi.edu.
© 2005 by The National Academy of Sciences of the USA
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